The arrest of bleeding involves the concerted action of various haemostatic pathways, which eventually lead to thrombus formation. Thrombi are depositions of blood constituents on the surface of the vessel wall, and are mainly composed of aggregated blood platelets and insoluble, cross-linked fibrin. Fibrin formation occurs by limited proteolysis of fibrinogen by thrombin. This enzyme is the final product of the coagulation cascade, a sequence of zymogen activations which occur at the surface of activated platelets and leukocytes, and of a variety of vascular cells (for review see K. G. Mann et al., Blood vol 76, 1990, pp 1-16).
Normally thrombus formation remains localized at sites of vascular injury, since platelet aggregation and adhesion is mediated by agonists associated with the growing thrombus, such as locally formed thrombin. Under certain pathological conditions however, the formation of these depositions does not remain limited to the site of injury. Thrombi then may occur in arteries and veins anywhere in the circulation, which may ultimately result in vessel obstruction and blood flow arrest. This provides the mechanism underlying a variety of thrombotic disorders, including deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation, peripheral arterial disease, myocardial infarction and stroke (J. F. Mustard et al., in: A. L. Bloom and D. P. Thomas (Eds.), Haemostasis and Thrombosis, 2nd edition, Churchill-Livingstone, Edinburgh, 1987, pp 503-526).
Numerous strategies have been developed for the treatment of thrombotic disorders. These antithrombotic therapies have in common that they are based on interference in the haemostatic system. This approach carries the inherent risk of bleeding, since the haemostatic system is no longer fully responsive to potential injury. Therefore, antithrombotic benefits are inevitably associated with antihaemostatic risks. In attempts to improve the benefit-to-risk ratio, novel antithrombotic agents are continuously being developed. The various antithrombotic strategies have been extensively reviewed elsewhere, and some are briefly summarized below to illustrate the various developments in this field. These include:
(a) General inhibitors of thrombin formation. One well established strategy consists of oral anticoagulant therapy employing vitamin K antagonists. This interferes in the biosynthesis of the so-called vitamin K-dependent coagulation factors, which include Factors VII, IX, X and prothrombin. This therapy thus is aspecific, as it affects both the extrinsic and the intrinsic coagulation pathway (see FIG. 1). Although widely used, oral anticoagulation requires intensive monitoring in order to reduce the bleeding risk associated with this therapy (see J. Hirsh et al., in: R. W. Colman et al. (Eds.), Hemostasis and Thrombosis, Basic Principles and Clinical Practice, 3rd edition, Lippincott, Philadelphia, 1994, pp 1567-1583). A similarly aspecific therapy consists of the administration of heparin. This compound accelerates the inactivation of a number of components, including thrombin and the activated forms of Factors IX and X (Factors IXa and Xa), by their natural inhibitor Antithrombin III. Over the past two decades, low molecular weight derivatives of heparin, which display an increased specificity for Factor Xa over thrombin inhibition, have been developed in an attempt to reduce the bleeding risk associated this therapy (see T. W. Barrowcliffe and D. P. Thomas, in: A. L. Bloom et al. (Eds.), Haemostasis and Thrombosis, 3rd edition, Churchill-Livingstone, Edinburgh, 1994, pp 1417-1438).
(b) Specific thrombin inhibitors. As thrombin is the key enzyme in platelet activation, fibrin formation, and activation of the cofactors Factors V and VIII (see FIG. 1), it may seem an attractive target for antithrombotic therapy. Numerous studies have been devoted to the small leech protein called hirudin, and to peptides derived thereof (J. M. Maraganore, Thromb. Haemostasis vol 70, 1993, pp 208-211). Although these components are more effective than for instance heparin in animal models of experimental thrombosis, clinical trials initially revealed an unexpectedly high frequency of bleeding, demonstrating that the thrombin-directed approach may produce a significant antihaemostatic effect, with a relatively unfavorable benefit-to-risk ratio (L. A. Harker, Biomedical Progress vol 8, 1995, 17-26).
(c) Specific Factor Xa inhibitors. Suppression of thrombin formation can effectively be achieved by inhibiting Factor Xa, which is the prothrombin activating enzyme in the coagulation cascade (see FIG. 1). Theoretically, this has the advantage of inhibiting vascular thrombus formation while still permitting a small, but haemostatically important amount of thrombin to be produced. Two naturally occurring peptide inhibitors of Factor Xa have recently been developed, the tick anticoagulant peptide (G. P. Vlasuk, Thromb. Haemostasis vol 70, 1993, pp 212-216) and antistasin, a leech anticoagulant (G. P. Tuszynsky et al., J. Biol. Chem. vol 262, 1987, pp 9718-9723). These polypeptides, and various oligopeptide derivatives thereof (N. Ohta et al., Thromb, Haemostasis vol 72, 1994, pp 825-830) may provide a more favorable benefit-to-risk ratio than agents with a broader specificity.
(d) Inhibitors of platelet activation and adhesion. Numerous studies have addressed this strategy, which has the theoretical advantage of specifically interrupting thrombin-dependent platelet recruitment at sites of vascular injury, while sparing the production of fibrin (see L. A. Harker et al., in: R. W. Colman et al. (Eds.), Hemostasis and Thrombosis, Basic Principles and Clinical Practice, 3rd edition, Lippincott, Philadelphia, 1994, pp 1638-1660). This strategy can be accomplished by various agents, including synthetic thrombin receptor antagonists, or monoclonal antibodies or peptides that interfere in the adhesion process. Although this approach seems particularly useful in arterial thrombosis, bleeding episodes still have been reported, suggesting that the specificity of this strategy may not have satisfactorily eliminated the antihaemostatic risk.
In summary, evaluation of current antithrombotic strategies in terms of antithrombotic benefits versus antihaemostatic risks reveals that the benefit-to risk ratio tends to be more favorable for strategies that interfere in one specific step rather than in a more general phase of the haemostatic system (L. A. Harker, Biomedical Progress vol 8, 1995, 17-26). Although the development of inhibitors specific for Factor Xa seems to be a promising improvement, this approach still blocks the common (intrinsic and extrinsic) pathway of thrombin generation (see FIG. 1), and thereby thrombin-dependent platelet activation as well. An urgent need therefore exists for more specific anti-thrombotic agents that inhibit one single haemostatic pathway, while leaving other pathways unaffected.
More selective inhibition of the haemostatic system should be achievable if agents would exist that exclusively interfere in the intrinsic pathway of Factor X activation. This would leave the extrinsic pathway intact, allowing the formation of small, but haemostatically important amounts of Factor Xa and thrombin. While the formation of the platelet plug associated with the initial phase of bleeding arrest thus would remain unaffected, the formation of larger amounts of thrombin and fibrin would be suppressed (see FIG. 1). A potentially successful strategy for achieving selective inhibition of the intrinsic coagulation pathway may consist of employing Factor VIII as a template for designing antagonist peptides which counteract its biological function. One attempt has been described which utilizes synthetic peptides mimicking the phospholipid-binding domain of Factor VIII (T. S. Zimmerman et al., International Patent Application, WO 90/15615, published Dec. 27, 1990). This region, which is located at the utmost C-terminus of the Factor VIII protein (the so-called C2-domain, see below), comprises a sequence that binds to specific phospholipids (phosphatidylserine) on the surface of activated platelets, leukocytes or vascular cells (P. A. Foster et al., Blood vol 75, 1990, pp 1999-2004). These phospholipid-binding peptides impede Factor VIII from participating in the blood coagulation process and as such may act as anticoagulants. However, because coagulation factors other than Factor VIII, including Factor V, Factor VII, Factor IX, Factor X and prothrombin (see FIG. 1) share the same requirement for phosphatidylserine-containing membranes (see K. G. Mann et al., Blood vol 76, 1990, pp 1-16), these phospholipid-binding peptides affect multiple steps in the coagulation system. Therefore, this approach lacks the desired specificity for the intrinsic coagulation pathway. Such selective inhibition should be achievable by using compounds which interfere in Factor VIII-Factor IX interaction, for instance by blocking the Factor VIII binding site on Factor IXa immediately after its formation during the haemostatic response. It would be even more preferable if such compounds would block the Factor VIII binding site on Factor IX prior to its conversion into Factor IXa, thus allowing prophylactic application as well. The design of such agents, however, until now has been hampered by a lack of knowledge concerning the molecular sites involved in Factor VIII(a)-Factor IX(a) interaction.
With regard to the binding sites involved in the inter-action between Factors VIII and IX, only a few reports have been published so far. These have focused on Factor VIII, which is the cofactor of Factor IXa in the Factor X activating complex (see FIG. 1). Factor VIII is synthesized as a single chain polypeptide of 2332 amino acids, with the typical domain structure A1-A2-B-A3-C1-C2 (G. A. Vehar et al., Nature vol 312, 1984, pp 337-342; J. J Toole et al., Nature vol 312, 1984, 342-347). Due to endoproteolytic processing, Factor VIII circulates in plasma as a heterodimeric complex of heavy and light chain. The light chain comprises amino acid residues 1649-2332, and contains the A3-C1-C2 domains. The heavy chain contains the domains A1-A2-B (residues 1-1648) and is heterogeneous due to limited proteolysis at a number of positions within the B-domain. Lenting et al. (J. Biol. Chem. vol 269, 1994, pp 7150-7155) have reported that Factor IXa specifically binds to the light chain of Factor VIII. Subsequently, the same investigators have reported similar studies employing Factor VIII light chain cleavage products, and concluded that the Factor IXa binding site should be located between residues 1722 and 2332 on the Factor VIII light chain (M. J. S. H. Donath et al., J. Biol. Chem. vol 270, 1995, pp 3648-3655). It has further been observed that the interaction between Factor IXa and Factor VIII light chain is inhibited by a monoclonal antibody, designated CLB-CAg A, which is directed against the Factor VIII A3-domain (P. J. Lenting et al., J. Biol. Chem. vol 269, 1994, pp 7150-7155), and more specifically against a region spanning the residues 1801-1823 (J. W. van de Loo et al., Annual Report 1992, Dr. Karl Landsteiner Foundation, Amsterdam, p 1).
Although these observations suggest that the Factor VIII A3-domain contributes to Factor IXa binding, other investigators have disclosed data that teach away from the Factor VIII light chain being a significant Factor IXa binding region. Fulcher et al. (Scientific Report 1992-1993, Scripps Research Institute, La Jolla, U.S.A., p 159) reported that a monoclonal antibody against the Factor VIII heavy chain region 701-750, or synthetic peptides derived from residues 698-710 or 698-712, inhibit Factor IXa-dependent functional assays. The same study reports that these peptides directly interact with Factor IXa, thus supporting the concept that the Factor VIII A2-domain region 698-710 represents a functionally important Factor IXa binding site (J. I. Jorquera et al., Circulation vol 86, 1992, p 685). Additional evidence for involvement of the Factor VIII heavy chain A2-domain in Factor IXa binding, albeit at a different site, has been reported by other investigators (P. J. Fay et al., J. Biol. Chem. vol 269, 1994, p 20522-20527). In the latter study, a synthetic peptide corresponding to the A2-domain residues 558-565 has been employed to identify this region as a Factor IXa interactive site.
In summary, a number of potential Factor IXa binding sites have been identified: at least two distinct sites within the A2-domain on the Factor VIII heavy chain, and one or more additional sites on the Factor VIII light chain region 1722-2332, presumably within the A3-domain. These divergent data imply that either multiple sites exist which are involved in Factor VIII binding, or that multiple sequences contribute to the formation of one single Factor IXa binding site. Thus, it remains difficult to conclusively identify a predominant site as appropriate template for the design of effective antihaemostatic agents. Moreover, prior disclosures have been limited to the interaction of Factor VIII with the enzyme Factor IXa. The non-activated proenzyme Factor IX, which may display significantly different binding requirements, has not been previously addressed.